4″-substituted-9-deoxo-9a-aza-9a homoerythromycin a derivative

ABSTRACT

The invention relates to a method of preparing compounds of the formula                    
     and to pharmaceutically acceptable salts thereof. The compounds of formula 1 are antibacterial agents that may be used to treat various bacterial and protozoa infections. The invention also relates to pharmaceutical compositions containing the compounds of formula 1 and to methods of treating bacterial protozoa infections by administering the compounds of formula 1. The invention also relates to methods of preparing the compounds of formula 1 and to intermediates useful in such preparation.

BACKGROUND OF THE INVENTION

This invention relates to novel C-4″ substituted derivatives of9-deoxo-9a-aza-9a-homoerythromycin A that are useful as antibacterialand antiprotozoa agents in mammals, including man, as well as in fishand birds. This invention also relates to pharmaceutical compositionscontaining the novel compounds and to methods of treating bacterialinfections and protozoa infections in mammals, fish and birds byadministering the novel compounds to mammals, fish and birds requiringsuch treatment.

Macrolide antibiotics are known to be useful in the treatment of a broadsprectrum of bacterial infections and protozoa infections in mammals,fish and birds. Such antibiotics include various derivatives oferythromycin A such as azithromycin which is commercially available andis referred to in U.S. Pat. Nos. 4,474,768 and 4,517,359, both of whichare incorporated herein by reference in their entirety. Likeazithromycin and other macrolide antibiotics, the novel macrolidecompounds of the present invention possess potent activity againstvarious bacterial infections and protozoa infections as described below.

SUMMARY OF THE INVENTION

The present invention relates to compounds of the formula

and to pharmaceutically acceptable salts thereof, wherein:

R¹ is H, hydroxy or methoxy;

R² is hydroxy;

R³ is C₁-C₁₀ alkyl, C₂-C₁₀ alkynyl, C₂-C₁₀ alkynyl, cyano,—CH₂S(O)_(n)R⁸ wherein n is an integer ranging from 0 to 2, —CH₂OR⁸,—CH₂N(OR⁹)R⁸, —CH₂NR⁸R¹⁵, —(CH₂)_(m)(C₈-C₁₀ aryl), or —(CH₂)_(m)(5-10membered heteroaryl), wherein m is an integer ranging from 0 to 4, andwherein the foregoing R³ groups are optionally substituted by 1 to 3 R¹⁶groups;

or R² and R³ are taken together to form an oxazolyl ring as shown below

R⁴ is H, —C(O)R⁹, —C(O)OR⁹, —C(O)NR⁹R¹⁰ or a hydroxy protecting group;

R⁵ is —SR⁸, —(CH₂)_(n)C(O)R⁸ wherein n is 0 or 1, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10membered heteroaryl), wherein m is an integer ranging from 0 to 4, andwherein the foregoing R⁵ groups are optionally substituted by 1 to 3 R¹⁶groups;

each R⁶ and R⁷ is independently H, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkyl,C₂-C₈ alkenyl, C₂-C₆ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl), or—(CH₂)_(m)(5-10 membered heteroaryl), wherein m is an integer rangingfrom 0 to 4;

each R⁸ is independently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, —(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)NR¹³R¹⁴ wherein q and r are eachindependently an integer ranging from 0 to 3 except q and r are not both0, —(CH₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10 membered hetercaryl),wherein m is an integer ranging from 0 to 4, and wherein the foregoingR⁸ groups, except H, are optionally substituted by 1 to 3 R¹⁶ groups;

or where R⁸ is as —CH₂NR⁸R¹⁵, R¹⁵ and R⁸ may be taken together to form a4-10 membered monocyclic or polycyclic saturated ring or a 5-10 memberedheteroaryl ring, wherein said saturated and heteroaryl rings optionallyinclude 1 or 2 heteroatoms selected from O, S and —N(R⁸)—, in additionto the nitrogen to which R¹⁵ and R⁸ are attached, said saturated ringoptionally includes 1 or 2 carbon-carbon double or triple bonds, andsaid saturated and heteroaryl rings are optionally substituted by 1 to 3R¹⁶ groups;

each R⁹ and R¹⁰ is independently H or C₁-C₆ alkyl;

each R¹¹, R¹², R¹³ and R¹⁴ is independently selected tom H, C₁-C₁₀alkyl, —(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 memberedheteroaryl), wherein m is an integer ranging from 0 to 4, and whereinthe foregoing R¹¹, R¹², R¹³ and R¹⁴ groups, except H, are optionallysubstituted by 1 to 3 R¹⁶ groups;

or R¹¹ and R¹³ are taken together to form —(CH₂)_(p)— wherein p is aninteger ranging from 0 to 3 such that a 4-7 membered saturated ring isformed that optionally includes 1 or 2 carbon-carbon double or triplebonds;

or R¹³ and R¹⁴ are taken together to form a 4-10 membered monocyclic orpolycyclic saturated ring or a 5-10 membered heteroaryl ring, whereinsaid saturated and heteroaryl rings optionally include 1 or 2heteroatoms selected from O, S and —N(R⁸)—, in addition to the nitrogento which R¹³ and R¹⁴ are attached, said saturated ring optionallyincludes 1 or 2 carbon-carbon double or triple bonds, and said saturatedand heteroaryl rings are optionally substituted by 1 to 3 R¹⁶ groups;

R¹⁵ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl, wherein theforegoing R¹⁵ groups are optionally substituted by 1 to 3 substituentsindependently selected from halo and —OR⁹;

each R¹⁶ is independently selected from halo, cyano, nitro,trifluoromethyl, azido, —C(O)R¹⁷, —C(O)OR¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷,—NR⁶C(O)R⁷, —C(O)NR⁶R⁷, —NR⁶R⁷, hydrdroxy, C₁-₆ alkyl, C₁-C₆ alkoxy,—(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein said aryl andheteroaryl subsituents are optionally substituted by 1 or 2 substituentsindependently selected from halo, cyano, nitro, trifluoromethyl, azido,—C(O)R¹⁷, —C(O)OR¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷, —NR⁶C(O)R⁷, —C(O)NR⁶R⁷,—NR⁶R⁷, hydroxy, C₁-C₆ alkyl, and C₁-C₆ alkoxy;

each R¹⁷ is independently selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 memberedheteroaryl), wherein m is an integer ranging from 0 to 4;

with the proviso that R⁸ is not H where R³ is ——CH₂S(O)_(n)R⁸.

Preferred compounds of formula 1 include those wherein R³ is hydroxy, R²is hydroxy, R³ is —CH₂NR¹⁵R⁸ or —CH₂SR⁸, and R⁴ is H.

Other preferred compounds of formula 1 include those wherein R³ ishydroxy, R² is hydroxy, R³ is —CH₂NR⁸R¹⁵, R⁴ is H, R¹⁵ and R⁸ are eachselected from H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl,wherein said R¹⁵ and R⁸ groups, except H, are optionally substituted by1 or 2 substituents independently selected from hydroxy, halo and C₁-C₆alkoxy. Specific preferred compounds having the foregoing generalstructure include those wherein R¹⁵ is either H or is selected from thefollowing groups from which R⁸ is also independently selected: methyl,ethyl, allyl, n-butyl, isobutyl, 2-methoxyethyl, cyclopentyl,3-methoxypropyl, 3-ethoxypropyl, n-propyl, isopropyl, 2-hydroxyethyl,cyclopropyl, 2,2,2-trifluoroethyl, 2-propynyl, sec-butyl, tert-butyl,and n-hexyl.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R³ is —CH₂NHR⁸, R⁴ is H, and R⁸ is—(CH₂)_(m)(C₆-C₁₀ aryl) wherein m is an integer ranging from 0 to 4.Specific preferred compounds having the foregoing general structureinclude those wherein R⁸ is phenyl or benzyl.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R³ is —CH₂NR¹⁵R⁸, R⁴ is H, and R¹⁵ and R⁸ aretaken together to form a saturated ring. Specific preferred compoundshaving the foregoing general structure include those wherein R⁶ and R⁸are taken together to form a piperidino, trimethyleneimino, ormorpholino ring.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R³ is —CH₂NR¹⁵R⁸, R⁴ is H, and R¹⁵ and R⁸ aretaken together to form a heteroaryl ring optionally substituted by 1 or2 C₁-C₆ alkyl groups. Specific preferred compounds having the foregoinggeneral structure include those wherein R¹⁵ and R⁸ are taken together toform a pyrrolidino, triazolyl, or imidazolyl ring wherein saidheteroaryl groups are optionally substituted by 1 or 2 methyl groups.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R³ is —CH₂SR⁸, R⁴ is H, and R⁸ is selected fromC₁-C₁₀ alkyl, C₂-C₁₀ alkynyl, wherein said R⁸ groups are optionallysubstituted by 1 or 2 substituents independently selected from hydroxy,halo and C₁-C₆ alkoxy. Specific preferred compounds having the foregoinggeneral structure include those wherein R³ is methyl, ethyl, or2-hydroxyethyl.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R⁴ is H, and R³ is selected from C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, wherein said R³ groups areoptionally substituted by 1 or 2 substituents independently selectedfrom hydroxy, —C(O)R¹⁷, —NR⁶R⁷, halo, cyano, azido, 5-10 memberedheteroaryl, and C₁-C₆ alkoxy. Specific preferred compounds having theforegoing general structure include those wherein R³ is methyl, allyl,vinyl, ethynyl, 1-methyl-1-propenyl, 3-methoxy-1-propynyl,3-dimethylamino-1-propynyl, 2-pyridylethynyl, 1-propynyl,3-hydroxy-1-propynyl, 3-hydroxy-1-propenyl, 3-hydroxypropyl,3methoxy-1-propenyl, 3-methoxypropyl, 1-propynyl, n-butyl, ethyl,propyl, 2-hydroxyethyl, formylmethyl, 6cyano-1-pentynyl,3dimethylamino-1-propenyl, or 3-dimethylaminopropyl.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R⁴ is H, and R³ is —(CH₂)_(m)(5-10 memberedheteroaryl) wherein m is an integer ranging from 0 to 4. Specificpreferred compounds having the foregoing general structure include thosewherein R³ is 2-thienyl, 2-pyridyl, 1-methyl-2-imidazolyl, 2-furyl, or1-methyl-2-pyrrolyl.

Other preferred compounds of formula 1 include those wherein R¹ ishydroxy, R² is hydroxy, R⁴ is H, and R³ is CH₂)_(m)(C₆-C₁₀ aryl) whereinm is an integer ranging from 0 to 4. Specific preferred compounds havingthe foregoing general structure include those wherein R³ is phenyl.

Specific compounds of formula 1 include those wherein R² and R³ aretaken together to form an oxazolyl ring as shown below

wherein R⁵ is as defined above.

Specific compounds of formula 1 include those wherein R³ is selectedfrom the following:

wherein X³ is O, S or —N(R¹⁵), and wherein the —OR⁹ group may beattached at any available carbon on the phenyl group.

The invention also relates to a pharmaceutical composition for thetreatment of a bacterial infection or a protozoa infection in a, mammal,fish, or bird which comprises a therapeutically effective amount of acompound of formula 1, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

The invention also relates to a method of treating a bacterial infectionor a protozoa infection in a mammal, fish, or bird which comprisesadministering to said mammal, fish or bird a therapeutically effectiveamount of a compound of formula 1 or a pharmaceutically acceptable saltthereof.

The term “treatment”, as used herein, unless otherwise indicated,includes the treatment or prevention of a bacterial infection orprotozoa infection as provided in the method of the present invention.

As used herein, unless otherwise indicated, the terms “bacterialinfection(s)” and “protozoa infection(s)” include bacterial infectionsand protozoa infections that occur in mammals, fish and birds as well asdisorders related to bacterial infections and protozoa infections thatmay be treated or prevented by administering antibiotics such as thecompounds of the present invention. Such bacterial infections andprotozoa infections, and disorders related to such infections, includethe following: pneumonia, otitis media, sinusitus, bronchitis,tonsillitis, and mastoiditis related to Infection by Streptococcuspneumoniae, Haemophilus influenzae, Moraxella catarrhalis,Staphylococcus aureus, or Peptostreptococcus spp.; pharynigitis,rheumatic fever, and glomerulonephritis related to infection byStreptococcus pyogenes, Groups C and G streptococci, Clostridiumdiptheriae, or Actinobacillus haemolyticum; respiratory tract infectionsrelated to infection by Mycoplasma pneumoniae, Legionella pneumophila,Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydiapneumoniae; uncomplicated skin and soft tissue infections, abscesses andosteomyelitis, and puerperal fever related to infection byStaphylococcus aureus, coagulase-positive staphylococci (i.e., S.epidermidis, S. hemolyticus, etc.), Streptococcus pyogenes,Streptococcus agalactiae, Streptococcal groups C-F (minute-colonystreptococci), viridans streptococci, Corynebacterium minutissimum,Clostridium spp., or Bartonella henselae; uncomplicated acute urinarytract infections related to infection by Staphylococcus saprophyticus orEntercoccus spp.; urethritis and cervicitis; and sexually transmitteddiseases related to infection by Chlamydia trachomatis, Haemophilusducreyi, Treponema pallidum, Ureaplasma urealyticum, or Neiserriagonorrheae; toxin diseases related to infection by S. aureus (foodpoisoning and Toxic shock syndrome), or Groups A, B, and C streptococci;ulcers related to infection by Helicobacter pylori; systemic febrilesyndromes related to infection by Borrelia recurrentis; Lyme diseaserelated to infection by Borrelia burgdodferi; conjunctivitis, keratitis,and dacrocystitis related to infection by Chlamydia trachomatis,Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H.influenzae, or Listeria spp.; disseminated Mycobacterium avium complex(MAC) disease related to infection by Mycobacterium avium, orMycobacterium intracellulare; gastroenteritis related to infection byCampylobacter jejuni; intestinal protozoa related to infection byCryptosporidium spp.; odontogenic infection related to infection byviridans streptococci; persistent cough related to infection byBordetella pertussis; gas gangrene related to infection by Clostridiumperfingens or Bacteroides spp.; and atherosclerosis related to infectionby Helicobacter pylori or Chlamydia pneumoniae. Bacterial infections andprotozoa infections and disorders related to such infections that may betreated or prevented in animals include the following: bovinerespiratory disease related to infection by P. haem., P. multocida,Mycoplasma bovis, or Bordetella spp.; cow enteric disease related toinfection by E. coli or protozoa (i.e., coccidia, cryptosporidia, etc.);dairy cow mastitis related to infection by Staph. aureus, Strep. uberis,Strep. agalactiae, Strep. dysgalactiae, Klebsiella spp.,Corynebacterium, or Enterococcus spp.; swine respiratory disease relatedto infection by A. pleuro., P. multocida, or Mycoplasma spp.; swineenteric disease related to infection by E. coli, Lawsoniaintracellularis, Salmonella, or Serpulina hyodyisinteriae; cow footrotrelated to infection by Fusobacterium spp.; cow metritis related toinfection by E. coli, cow hairy warts related to infection byFusobacterium necrophorum or Bacteroides nodosus; cow pinkeye related toinfection by Moraxella bovis; cow premature abortion related toinfection by protozoa (i.e. neosporium); urinary tract infection in dogsand cats related to infection by E. coli; skin and soft tissueinfections in dogs and cats related to infection by Staph. epidermidis,Staph. intermedius, coagulase neg. Staph. or P. multocida; and dental ormouth infections in dogs and cats related to infection by Alcaligenesspp., Bacteroides spp., Clostridium spp., Enterobacter spp.,Eubacterium, Peptostreptococcus, Porphyromonas, or Prevotella. Otherbacterial infections and protozoa infections and disorders related tosuch infections that may be treated or prevented in accord with themethod of the present invention are referred to in J. P. Sanford et al.,“The Sanford Guide To Antimicrobial Therapy,” 26th Edition,(Antimicrobial Therapy, Inc., 1996).

The present invention also relates to a method of preparing the abovecompound of formula 1, or a pharmaceutically acceptable salt thereof,wherein R³ is —CH₂S(O)_(n)R⁸, —CH₂OR⁸ or —CH₂NR⁸R¹⁵, wherein n, R¹⁵ andR⁸ are as defined above with the proviso that R⁸ is not H where R³ is—CH₂S(O)_(n)R⁸, which comprises treating a compound of the formula

wherein R¹ and R⁴ are as defined above, with a compound of the formulaHSR⁸, HOR⁸ or HNR¹⁵R⁸, wherein n, R¹⁵ and R⁸ are as defined above,optionally followed by oxidation of the —SR⁸ substituent to form —S(O)R⁸or (O)₂R⁸.

In a further aspect of the above process of preparing the compound offormula 1, or a pharmaceutically acceptable salt thereof, the abovecompound of formula 5 is prepared by treating a compound of the formula

wherein R¹ and R⁴ are as defined above, with (CH₃)₃S(O)_(n)X², wherein nis 0 or 1 and X² is halo, —BF₄ or —PF₆, preferably iodo or —BF₄, in thepresence of a base such as as potassium tert-butoxide, sodiumtert-butoxide, sodium ethoxide, sodium hydride,1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene,1,5-diazabicylo[4.3.0]non-5ene, potassium hexamethyldisilazide (KHMDS),potassium ethoxide, or sodium methoxide, preferably KHMDS or asodium-containing base such as sodium hydride.

The present invention also relates to the above compounds of formulas 4and 5 which, as indicated above, are useful in the preparation of theabove compounds of formula 1 and pharmaceutically acceptable saltsthereof.

The term “hydroxy protecting group”, as used herein, unless otherwiseindicated, includes acetyl, benzyloxycarbonyl, and various hydroxyprotecting groups familiar to those skilled in the art include thegroups referred to in T. W. Greene, P. G. M. Wuts, “Protective Groups InOrganic Synthesis,” (J. Wiley & Sons, 1991).

The term “halo”, as used herein, unless otherwise indicated, includesfluoro, chloro, bromo or iodo.

The term “alkyl”, as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight, cyclic orbranched moieties, or mixtures thereof. It is to be understood thatwhere cyclic moieties are intended, at least three carbons in said alkylmust be present. Such cyclic moieties include cyclopropyl, cyclobutyland cyclopentyl.

The term “alkoxy”, as used herein, unless otherwise indicated, includes—O-alkyl groups wherein alkyl is as defined above.

The term “aryl”, as used herein, unless otherwise indicated, includes anorganic radical derived from an aromatic hydrocarbon by removal of onehydrogen, such as phenyl or naphthyl.

The term “5-10 membered heteroaryl”, as used herein, unless otherwiseindicated, includes aromatic heterocyclic groups containing one or moreheteroatoms each selected from O, S and N, wherein each heterocyclicgroup has from 5 to 10 atoms in its ring system. Examples of suitable5-10 membered heteroaryl groups include pyridinyl, imidazolyl,pyrimidinyl, pyrazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl andthiazolyl.

The phrase “pharmaceutically acceptable salt(s)”, as used herein, unlessotherwise indicated, includes salts of acidic or basic groups which maybe present in the compounds of the present invention. The compounds ofthe present invention that are basic in nature are capable of forming awide variety of salts with various inorganic and organic acids. Theacids that may be used to prepare pharmaceutically acceptable acidaddition salts of such basic compounds of the present invention arethose that form non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions, such as the hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, isonicotinate, acetate, lactate, salicylate, citrate, acidcitrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylenen-bis-(2-hydroxy-3-naphthoate)]salts. The compounds of thepresent invention that include an amino moiety may form pharmaceuticallyacceptable salts with various amino acids, in addition to the acidsmentioned above.

Those compounds of the present invention that are acidic in nature arecapable of forming base salts with various pharmacologically acceptablecations. Examples of such salts include the alkali metal or alkalineearth metal salts and, particularly, the calcium, magnesium, sodium andpotassium salts of the compounds of the present invention.

Certain compounds of the present invention may have asymmetric centersand therefore exist in different enantiomeric and diastereomic forms.This invention relates to the use of all optical isomers andstereoisomers of the compounds of the present invention, and mixturesthereof, and to all pharmaceutical compositions and methods of treatmentthat may employ or contain them.

The present invention includes the compounds of the present invention,and the pharmaceutically acceptable salts thereof, wherein one or morehydrogen, carbon or other atoms are replaced by isotopes thereof. Suchcompounds may be useful as research and diagnostic tools in metabolismpharmacokinetic studies and in binding assays.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of of the present invention may be prepared according toSchemes 1-3 below and the description that follows.

The compounds of the present invention are readily prepared. Referringto the Schemes illustrated above, the starting compound of formula 2 maybe prepared according to one or more methods familiar to those skilledin the art including the synthetic methods described in U.S. Pat. Nos.4,474,768 and 4,517,359, referred to above. In step 1 of Scheme 1, theC-2′ hydroxy group may be selectively protected by treating the compoundof formula 2 with one equivalent of acetic anhydride in dichloromethanein the absence of external base to provide the compound of formula 3wherein R⁴ is acetyl. The acetyl protecting group may be removed bytreating the compound of formula 3 with methanol at 23-65° C. for 10-48hours. The C-2′ hydroxy may also be protected with other hydroxyprotecting groups familiar to those skilled in the art, such as thebenzyloxycarbonyl (Cbz) group. The C-9a amino group may also requireprotection before further synthetic modifications are performed.Suitable protecting groups for the amino moiety are Cbz andt-butyloxycarbonyl (Boc) groups. To protect the C-9a amino group, themacrolide may be treated with t-butyl dicarbonate in anhydroustetrahydrofuran (THF) or benzyloxycarbonyl N-hydroxysuccinimide ester orbenzylchloroformate to protect the amino group as its t-butyl or benzylcarbamate. Both the C-9a amino and C-2′ hydroxy may be selectivelyprotected with the Cbz group in one step by treating the compound offormula 2 with benzylchloroformate in THF and water. The Boc group maybe removed by acid treatment and the Cbz group may be removed byconventional catalytic hydrogenation. In the following description, itis assumed that the C-9a amino moiety and the C-2′ hydroxy group areprotected and deprotected as would be deemed appropriate by thoseskilled in the art.

In step 2 of Scheme 1, the C-4″ hydroxy group of the compound of formula3 is oxidized to the corresponding ketone by methods familiar to thoseskilled in the art, including one or more methods described in theJournal of Antibiotics, 1988, pages 1029-1047. For example, the ketoneof formula 4 may be prepared with DMSO and an appropriate activatingagent. Typical reaction conditions for the oxidation include: (a)Moffatt oxidation which employsN-ethyl-N′-(N,N-dimethylaminopropyl)carbodiimide and DMSO in thepresence of pyridinium trifluoroacetate; or (b) Swern oxidation in whichoxalyl chloride and DMSO in CH₂Cl₂ is followed by the addition oftriethylamine or alternatively trifluoracetic anhydride and DMSO inCH₂Cl₂ is followed by the addition of triethylamine. In step 3 of Scheme1, the compound of formula 4 is treated with R³MgX¹ or R³-Li andMg(X¹)₂, wherein X¹ is a halide such as chloro or bromo, in a solventsuch as THF, ethylene glycol dimethyl ether (DME), diisopropyl ether,toluene, diethyl ether, or tetramethylethylenediamine (TMEDA), hexanes,or a mixture of two or more of the foregoing solvents, preferably anether solvent, at a temperature ranging from about −78° C. to about roomtemperature (20-25° C.), to provide the compound of formula 1 wherein R²is hydroxy and R¹, R³ and R⁴ are as defined above.

Scheme 2 illustrates the preparation of compounds of formula 1 throughuse of an epoxide intermediate. In step 1 of Scheme 2, the compound offormula 5 may be generated by two methods. In one method (Method A), thecompound of formula 4 is treated with (CH₃)₃S(O)X², wherein X² is halo,—BF, or —PF₆, preferably iodo, in the presence of a base such as aspotassium tert-butoxide, sodium tert-butoxide, sodium ethoxide, sodiumhydride, 1,1,3,3-tetramethylguanidine,1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene,potassium ethoxide, or sodium methoxide, preferably a sodium-containingbase such as sodium hydride, in a solvent such as THF, an ether solvent,dimethylformamide (DMF), or methyl sulfoxide (DMSO), or a mixture of twoor more of the foregoing solvents, at a temperature within the range ofabout 0° C. to about 60° C., the compound of formula 5 is generated inwhich the following configuration of the epoxide moiety may predominate

In a second method (Method B), the compound of formula 4 is treated with(CH₃)₃SX², wherein X² is halo, —BF₄ or —PF₆, preferably —BF₄, in thepresence of a base such as as potassium tert-butoxide, sodium ethoxide,sodium tert-butoxide, sodium hydride, 1,1,3,3-tetramethylguanidine,1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene,potassium ethoxide, potassium hexamethyldisilazide (KHMDS) or sodiummethoxide, preferably KHMDS, in a solvent such as THF, an ether solvent,DMF, or DMSO, or a mixture of two or more of the foregoing solvents, ata temperature within the range of about −78° C. to about 60° C., toprovide the compound of formula 1 in which the following configurationof the epoxide moiety predominates

In step 2 of Scheme 2, the compound of formula 5 may be converted to acompound of formula 1 wherein R² is hydroxy and R³ is a group that isattached to the C-4″ carbon through a methylene group, such as where R³is —CH₂NR¹⁵R⁸ or —CH₂S(O)_(n)R⁸ wherein n, R¹⁵ and R⁸ are as definedabove. To prepare a compound of formula 1 wherein R³ is —CH₂NR¹⁵R⁸, thecompound of formula 5 may be treated with a compound of the formulaHNR¹⁵R⁸, wherein R¹⁵ and R⁸ are as defined above, in the absence orpresence of a polar solvent such as water, methanol, or THF, or amixture of the foregoing solvents, at a temperature ranging from aboutroom temperature to about 100° C., preferably about 60° C., optionallyin the presence of a halide reagent such as potassium iodide, lithiumperchlorate, magnesium perchlorate, lithium tetrafluoroborate,pyridinium hydrochloride, or a tetraalkylammonium halide reagent such astetrabutylammonium iodide. To prepare a compound of formula 1 wherein R³is —CH₂S(O)_(n)R⁸ wherein n and R⁸ are as defined above, the compound offormula 5 may be treated with a compound of the formula HSR⁸ in thepresence of K₂CO₃, Kl, or sodium methoxide, in an aromatic solvent suchas methanol, benzene or toluene at a temperature ranging from about roomtemperature to about 120° C. As appropriate, the sulfur moiety may beoxidized to —SO— or —SO₂— according to methods familiar to those skilledin the art. To prepare a compound of formula 1 wherein R³ is —CH₂SR⁸ andR⁸ is —(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)NR¹³R¹⁴, wherein the substituents ofsaid R⁸ group are as defined above, the compound of formula 5 may betreated with a compound of the formulaHS—(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)-NPhth, wherein NPhth representsphthalimido, and potassium iodide to provide the compound of formula 1wherein R³ is —CH₂S(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)NH₂, after removal of thephthalimido moiety, which may be further modified as necessary. Usingthe same or an analogous method, a compound of formula 1 wherein R³ is—CH₂NR¹⁵R⁸ and R⁸ is —(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)—NR¹³R¹⁴ may be preparedby treating the compound of formula 5 with either a compound of theformula HNR⁹—(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)—NR¹³R¹⁴ or a compound or theformula H₂N—(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)—NH₂ followed by reductivealkylation of the nitrogen atoms. Using the same or an analogous method,a compound of formula 1 wherein R³ is —CH₂OR⁸ and R⁸ is as defined abovemay be prepared by treating a compound of formula 5 with a compound ofthe formula HOR⁸.

Scheme 3 illustrates the preparation of compounds of formula 1 in whichR² and R³ are taken together to form an oxazolyl moiety. In step 1 ofScheme 3, the compound of formula 5 is treated with sodium azide in thepresence of NH₄Cl in methanol or water, or a mixture of the twosolvents, at a temperature ranging from about 0° C. to about 100° C.,preferably about 80° C., to provide the compound of formula 6. In step 2of Scheme 3, the compound of formula 6 may be converted to thecorresponding amine of formula 7 via conventional catalytichydrogenation. Preferably, such hydrogenation is done using Pd (10% oncarbon) powder under an H₂ atmosphere (1 atm). The resulting amine offormula 7 may be converted to various compounds of formula 1 wherein R³is —CH₂NR¹⁵R⁸ using conventional synthetic methods such as reductiveamination.

In step 3 of Scheme 3, the compound of formula 7 may be converted to thecompound of formula 1 wherein R² and R³ are taken together as shown bytreating the compound of formula 7 with a compound of formula R⁵—CN,R⁵—═N(OCH₃), R⁵—C═N(OC₂H₅), R⁵—C(O)Cl, or R⁵—CO₂H, wherein R⁵ is asdefined above, except it is not NH₂, in the presence or absence of anacid, such as HCl, or a Lewis acid, such as ZnCl₂ or BF₄Et₃O, or a base,such as NaOH or TEA, in a solvent such as THF, a chlorohydrocarbon (suchas CH₂Cl₂ or chlorobenzene), at a temperature ranging from about roomtemperature to reflux. In the alternative, the compound of formula 7 mayproceed as indicated in steps 4 and 5 of Scheme 3. In step 4 of Scheme3, the compound of formula 7 is treated with thiocarbonyldiimidazole inmethylene chloride at a temperature ranging from about 0° C. to roomtemperature to provide the compound of formula 13. In step 5 of Scheme3, the compound of formula 13 is treated with R⁵—X¹, wherein X¹ is ahalide such as bromo or iodo, and a base such as sodium methoxide in asolvent such as methanol or acetone, or a mixture of the two solvents,at a temperature ranging from about 0° C. to room temperature.

The compounds of the present invention may have asymmetric carbon atomsand therefore exist in different enantiomeric and diastereomeric forms.Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods known to those skilled in the art, for example, bychromatography or fractional crystallization. Enantiomers may beseparated by converting the enantiomeric mixtures into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. The use of all such isomers, including diastereomermixtures and pure enantiomers, are considered to be part of the presentinvention.

The compounds of the present invention that are basic in nature arecapable of forming a wide variety of different salts with variousinorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to mammals, it is oftendesirable in practice to initially isolate the compound of the presentinvention from the reaction mixture as a pharmaceutically unacceptablesalt and then simply convert the latter back to the free base compoundby treatment with an alkaline reagent and subsequently convert thelatter free base to a pharmaceutically acceptable acid addition salt.The acid addition salts of the base compounds of this invention arereadily prepared by treating the base compound with a substantiallyequivalent amount of the chosen mineral or organic acid in an aqueoussolvent medium or in a suitable organic solvent, such as methanol orethanol. Upon careful evaporation of the solvent, the desired solid saltis readily obtained. The desired salt can also be precipitated from asolution of the free base in an organic solvent by adding to thesolution an appropriate mineral or organic acid.

Those compounds of the present invention that are acidic in nature arecapable of forming base salts with various cations. For compounds thatare to be administered to mammals, fish or birds such salts must bepharmaceutically acceptable. Where a pharmaceutically acceptable salt isrequired, it may be desirable to initially isolate the compound of thepresent invention from the reaction mixture as a pharmaceuticallyunacceptable salt and then simply convert the latter to apharmaceutically acceptable salt in a process analogous to thatdescribed above relating to the conversion of pharmaceuticallyunacceptable acid addition salts to pharmaceutically acceptable salts.Examples of base salts include the alkali metal or alkaline-earth metalsalts and particularly the sodium, amine and potassium salts. Thesesalts are all prepared by conventional techniques. The chemical baseswhich are used as reagents to prepare the pharmaceutically acceptablebase salts of this invention are those which form non-toxic base saltswith the acidic compounds of the present invention. Such non-toxic basesalts include those derived from such pharmacologically acceptablecations as sodium, potassium, calcium, magnesium, various amine cations,etc. These salts can easily be prepared by treating the correspondingacidic compounds with an aqueous solution containing the desiredpharmacologically acceptable bases with cations such as sodium,potassium, calcium, magnesium, various amine cations, etc., and thenevaporating the resulting solution to dryness, preferably under reducedpressure. Alternatively, they may also be prepared by mixing loweralkanolic solutions of the acidic compounds and the desired alkali metalalkoxide together, and then evaporating the resulting solution todryness in the same manner as before. In either case, stoichiometricquantities of reagents are preferably employed in order to ensurecompleteness of reaction and maximum yields of the desired finalproduct.

The antibacterial and antiprotozoa activity of the compounds of thepresent invention against bacterial and protozoa pathogens isdemonstrated by the compound's ability to inhibit growth of definedstrains of human (Assay I) or animal (Assays II and III) pathogens.

Assay I

Assay I, described below, employs conventional methodology andinterpretation criteria and is designed to provide direction forchemical modifications that may lead to compounds that circumventdefined mechanisms of macrolide resistance. In Assay I, a panel ofbacterial strains is assembled to include a variety of target pathogenicspecies, including representatives of macrolide resistance mechanismsthat have been characterized. Use of this panel enables the chemicalstructure/activity relationship to be determined with respect topotency, spectrum of activity, and structural elements or modificationsthat may be necessary to obviate resistance mechanisms. Bacterialpathogens that comprise the screening panel are shown in the tablebelow. In many cases, both the macrolide-susceptible parent strain andthe macrolide-resistant strain derived from it are available to providea more accurate assessment of the compound's ability to circumvent theresistance mechanism. Strains that contain the gene with the designationof ermA/ermB/ermC are resistant to macrolides, lincosamides, andstreptogramin B antibiotics due to modifications (methylation) of 23SrRNA molecules by an Erm methylase, thereby generally prevent thebinding of all three structural classes. Two types of macrolide effluxhave been described; msrA encodes a component of an efflux system instaphylococci that prevents the entry of macrolides and streptograminswhile mefA/E encodes a transmembrane protein that appears to efflux onlymacrolides. Inactivation of macrolide antibiotics can occur and can bemediated by either a phosphorylation of the 2′-hydroxyl (mph) or bycleavage of the macrocyclic lactone (esterase). The strains may becharacterized using conventional polymerase chain reaction (PCR)technology and/or by sequencing the resistance determinant. The use ofPCR technology in this application is described in J. Sutcliffe et al.,“Detection Of Erythromycin-Resistant Determinants By PCR”, AntimicrobialAgents and Chemotherapy, 40(11), 2562-2566 (1996). The assay isperformed in microtiter trays and interpreted according to PerformanceStandards for Antimicrobial Disk Susceptibility Tests—Sixth Edition:Approved Standard, published by The National Committee for ClinicalLaboratory Standards (NCCLS) guidelines; the minimum inhibitoryconcentration (MIC) is used to compare strains. Compounds are initiallydissolved in dimethylsulfoxide (DMSO) as 40 mg/ml stock solutions.

Strain Designation Macrolide Resistance Mechanism(s) Staphylococcusaureus 1116 susceptible parent Staphylococcus aureus 1117 ermBStaphylococcus aureus 0052 susceptible parent Staphylococcus aureus 1120ermC Staphylococcus aureus 1032 msrA, mph, esterase Staphylococcushemolyticus 1006 msrA, mph Streptococcus pyogenes 0203 susceptibleparent Streptococcus pyogenes 1079 ermB Streptococcus pyogenes 1062susceptible parent Streptococcus pyogenes 1061 ermB Streptococcuspyogenes 1064 ermB Streptococcus agalactiae 1024 susceptible parentStreptococcus agalactiae 1023 ermB Streptococcus pneumoniae 1016susceptible Streptococcus pneumoniae 1046 ermB Streptococcus pneumoniae1095 ermB Streptococcus pneumoniae 1175 mefE Streptococcus pneumoniae0085 susceptible Haemophilus influenzae 0131 susceptible Moraxeilacatarrhalis 0040 susceptible Moraxella catarrhalis 1055 erythromycinintermediate resistance Escherichia coli 0266 susceptible

Assay II is utilized to test for activity against Pasteurella multocidaand Assay III is utilized to test for activity against Pasteurellahaemolytica.

Assay II

This assay is based on the liquid dilution method in microliter format Asingle colony of P. multocida (strain 59A067) is inoculated into 5 ml ofbrain heart infusion (BHI) broth. The test compounds are prepared bysolubilizing 1 mg of the compound in 125 μl of dimethylsulfoxide (DMSO).Dilutions of the test compound are prepared using uninoculated BHIbroth. The concentrations of the test compound used range from 200 μg/mlto 0.098 μg/ml by two-fold serial dilutions. The P. multocida inoculatedBHI is diluted with uninoculated BHI broth to make a 10⁴ cell suspensionper 200 μl. The BHI cell suspensions are mixed with respective serialdilutions of the test compound, and incubated at 37° C. for 18 hours.The minimum inhibitory concentration (MIC) is equal to the concentrationof the compound exhibiting 100% inhibition of growth of P. multocida asdetermined by comparison with an uninoculated control.

Assay III

This assay is based on the agar dilution method using a SteersReplicator. Two to five colonies isolated from an agar plate areinoculated into BHI broth and incubated overnight at 37° C. with shaking(200 rpm). The next morning, 300 μl of the fully grown P. haemolyticapreculture is inoculated into 3 ml of fresh BHI broth and is incubatedat 37° C. with shaking (200 rpm). The appropriate amounts of the testcompounds are dissolved in ethanol and a series of two-fold serialdilutions are prepared. Two ml of the respective serial dilution ismixed with 18 ml of molten BHI agar and solidified. When the inoculatedP. haemolytica culture reaches 0.5 McFarland standard density, about 5μl of the P. haemolytica culture is inoculated onto BHI agar platescontaining the various concentrations of the test compound using aSteers Replicator and incubated for 18 hours at 37° C. Initialconcentrations of the test compound range from 100-200 μg/ml. The MIC isequal to the concentration of the test compound exhibiting 100%inhibition of growth of P. haemolytica as determined by comparison withan uninoculated control.

The in vivo activity of the compounds of formula (I) can be determinedby conventional animal protection studies well known to those skilled inthe art, usually carried out in mice.

Mice are allotted to cages (10 per cage) upon their arrival, and allowedto acclimate for a minimum of 48 hours before being used. Animals areinoculated with 0.5 ml of a 3×10³ CFU/ml bacterial suspension (P.multocida strain 59A006) intraperitoneally. Each experiment has at least3 non-medicated control groups including one infected with 0.1×challenge dose and two infected with 1× challenge dose; a 10× challengedata group may also be used. Generally, all mice in a given study can bechallenged within 30-90 minutes, especially if a repeating syringe (suchas a Cornwall® syringe) is used to administer the challenge. Thirtyminutes after challenging has begun, the first compound treatment isgiven. It may be necessary for a second person to begin compound dosingif all of the animals have not been challenged at the end of 30 minutes.The routes of administration are subcutaneous or oral doses.Subcutaneous doses are administered into the loose skin in the back ofthe neck whereas oral doses are given by means of a feeding needle. Inboth cases, a volume of 0.2 ml is used per mouse. Compounds areadministered 30 minutes, 4 hours, and 24 hours after challenge. Acontrol compound of known efficacy administered by the same route isincluded in each test. Animals are observed daily, and the number ofsurvivors in each group is recorded. The P. multocida model monitoringcontinues for 96 hours (four days) post challenge.

The PD₅₀ is a calculated dose at which the compound tested protects 50%of a group of mice from mortality due to the bacterial infection whichwould be lethal in the absence of drug treatment.

The compounds of formula 1, and the pharmaceutically acceptable saltsthereof (hereinafter “the active compounds”), may be adminstered throughoral, parenteral, topical, or rectal routes in the treatment ofbacterial and protozoa infections. In general, these compounds are mostdesirably administered in dosages ranging from about 0.2 mg per kg bodyweight per day (mg/kg/day) to about 200 mg/kg/day in single or divideddoses (i.e., from 1 to 4 doses per day), although variations willnecessarily occur depending upon the species, weight and condition ofthe subject being treated and the particular route of administrationchosen. However, a dosage level that is in the range of about 4mg/kg/day to about 50 mg/kg/day is most desirably employed. Variationsmay nevertheless occur depending upon the species of mammal, fish orbird being treated and its individual response to said medicament, aswell as on the type of pharmaceutical formulation chosen and the timeperiod and interval at which such administration is carried out. In someinstances, dosage levels below the lower limit of the aforesaid rangemay be more than adequate, while in other cases still larger doses maybe employed without causing any harmful side effects, provided that suchlarger doses are first divided into several small doses foradministration throughout the day.

The active compounds may be administered alone or in combination withpharmaceutically acceptable carriers or diluents by the routespreviously indicated, and such administration may be carried out insingle or multiple doses. More particularly, the active compounds may beadministered in a wide variety of different dosage forms, i.e., they maybe combined with various pharmaceutically acceptable inert carriers inthe form of tablets, capsules, lozenges, troches, hard candies, powders,sprays, creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the active compounds are present in such dosageforms at concentration levels ranging from about 5.0% to about 70% byweight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active compound may be combined with various sweetening or flavoringagents, coloring matter or dyes, and, if so desired, emulsifying and/orsuspending agents as well, together with such diluents as water,ethanol, propylene glycol, glycerin and various like combinationsthereof.

For parenteral administration, solutions of an active compound in eithersesame or peanut oil or in aqueous propylene glycol may be employed. Theaqueous solutions should be suitably buffered (preferably pH greaterthan 8) if necessary and the liquid diluent first rendered isotonic.These aqueous solutions are suitable for intravenous injection purposes.The oily solutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques will known to those skilled in the art.

Additionally, it is also possible to administer the active compounds ofthe present invention topically and this may be done by way of creams,jellies, gels, pastes, patches, ointments and the like, in accordancewith standard pharmaceutical practice.

For administration to animals other than humans, such as cattle ordomestic animals, the active compounds may be administered in the feedof the animals or orally as a drench composition.

The active compounds may also be adminstered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The active compounds may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenyl, polyhydroxyethylaspartamide-phenol, orpolyethyleneoxide-polylysine substituted with palmitoylresidues.Furthermore, the active compounds may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyglycolic acid, copolymers ofpolylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

The following Examples further illustrate the method and intermediatesof the present invention. It is to be understood that the presentinvention is not limited to the specific details of the Examplesprovided below.

TABLE 1 The compounds of Examples 1-32 have the general formula 8 belowwith the R substituents indicated in the table below. The compounds wereprepared as described in Preparations 1-7 below. In the table, the yieldand mass spectra (“Mass Spec”) data apply to the final product.

Example R Substituent Preparation Yield Mass Spec 1 n-butylamino 1 48%820 2 2-methoxyethylamino 1 52% 822 3 piperidino 1 61% 832 4 morpholino1 39% 834 5 t-butylamino 1 23% 821 6 benzylamino 1 34% 854 7cyclopentylamino 2 23% 832 8 propylamino 2 11% 806 9 anilino 1 21% 84110 2-methoxypropylamino 1 46% 835 11 azido 3 46% 790 12 hexylamino 1 56%847 13 3-ethoxypropylamino 1 52% 851 14 diethylamino 2 53% 821 15N-methylbutylamino 1 76% 835 16 N-methylpropylamino 2 59% 819 17ethylamino 5 18% 792 18 cyclopropylamino 2 50% 804 19 ethylmethylamino 292% 806 20 2,2,2-trifluoroethylamino 2 67% 846 21 allylamino 1 59% 80422 2-hydroxyethylthio 6 44% 826 23 dimethylamino 1 71% 793 24imidazol-1-yl 4 42% 815 25 bis(2-hydroxyethyl)amino 7 21% 853 26pyrrolidino 2 40% 818 27 2-hydroxy-ethylmethylamino 2 23% 822 281,2,3-triazol-1-yl 4 69% 817 29 2-propynylamino 2 51% 802 302-methylimidazol-1-yl 4 14% 829 31 diallylamino 2 29% 844 321,2,4-triazol-1-yl 4 34% 816

Preparation Methods for Table 1

With reference to the Scheme illustrated above, the compound of formula11 wherein R is H and R⁴ is H (25 g (34.01 mmol, 1.0 equiv)) was mixedin a solution with phenol red in 250 mL THF and 125 mL water. To thispink solution was slowly added 29 mL (204.1 mmol, 6.0 equiv)benzylchloroformate and 2N NaOH to keep the solution basic. The reactionwas allowed to stir at room temperature overnight. The reaction mixturewas concentrated to remove the THF and the aqueous phase was adjusted tothe pH of 9.5 and extracted 3×500 mL EtOAc. The combined organic layerswere washed with 500 mL brine and then dried over Na₂CO₃. Filtration,concentration of the filtrate, and drying afforded a crude material.Further purification was done by column chromatography (100% CH₂Cl₂ toremove impurities and then 5% MeOH/CH₂Cl₂ to remove product) to yield32.6 g (96%) of a yellowish solid which was the compound of formula 11wherein R and R⁴ were both Cbz (MS (FAB) m/z 1003). 32.6 g (32.49 mmol,1.0 equiv) of this product was dissolved in 216.6 mL CH₂Cl₂ and 27.3 mLof DMSO. To this solution, 21.2 g (110.5 mmol, 3.4 equiv) of EDC and24.1 g (124.8 mmol, 3.8 equiv) PTFA were added. After stirring overnightthe reaction was quenched with 150 mL of water and the pH was adjustedto 9.5 with the addition of 2N NaOH. The organic layer was extracted3×150 mL CH₂Cl₂ and dried over Na₂SO₄. Filtration, concentration of thefiltrate, and drying afforded a crude yellow oil. Further purificationon a silica gel column (2% MeOH/CHCl₃) to give 25.6 g (79%) of ayellowish solid which was the compound of formula 12 wherein both R andR⁴ were Cbz

14 g (13.98 mmol, 1.0 equiv) of the compound of formula 12 prepared asdescribed above was dissolved in 1 L of 2-propanol and to this was added14 g of 10% Pd/C. The mixture was hydrogenated at 50 psi for three days.14 g of 10% Pd/C was added to the reaction and allowed to stir foranother day. This was repeated again and stirred for another day. Thecatalyst was removed by filtration through Celite and a minimal wash of2-propanol to yield 4.8 g (47%) of the compound of formula 12 whereinboth R and R⁴ were H (MS (APCi) m/z 734).

6.7 g (169.17 mmol, 6.2 equiv) of NaH (60% in oil dispersion) was washedtwice with 150 mL hexanes to remove the mineral oil. The solid wasdiluted in 335 mL of DMSO and 38.4 g (174.62 mmol, 6.4 equiv) of Me₃SOIwas added in three portions. The soution was stirred for an hour oruntil it turned dear. 20 g (27.29 mmol, 1.0 equiv) of the compound offormula 12 wherein both R and R⁴ were H was dissolved in 200 mL of THF.The ketone was transferred via cannula to the reaction flask and allowedto stir for 20 minutes. The reaction was quenched with 500 mL saturatedNaHCO₃, extracted 4×500 mL EtOAc, and dried over Na₂SO₄. Filtration,concentration of the filtrate, and drying gave the crude oil. Furtherpurification on 750 g of silica gel (5% MeOH/CHCl₃, 0.3% NH₄OH) afforded8.8 g (43%) of a white solid which was the compound of formula 13 (MS(TS) m/z 747).

Preparation 1

250-500 mg of the above compound of formula 13 was dissolved in 1-2 mLof an amine corresponding to the R substituent specified in Table 1. Acatalytic amount (20 mg) of pyridinium hydrochloride was added and thesolution was heated to 50-85° C. for one to seven days. The reaction wasworked up by quenching with 50 mL saturated NaHCO₃, extracted with 3×50mL CH₂Cl₂, and dried over Na₂SO₄. Filtration, concentration of thefiltrate, and drying gave a crude oil or solid. Further purification ona silica gel column (2-4% MeOH/CHCl₃, 0.2% NH₄OH) afforded the finalproduct.

Preparation 2

250-500 mg of the above compound of formula 13 was dissolved in 1-2 mLof an amine corresponding to the R substituent specified in Table 1 in asealed tube. A catalytic amount (20 mg) of pyridinium hydrochloride wasadded and the solution was heated to 50-75° C. for one to five days. Thereaction was worked up by quenching with 50 mL saturated NaHCO₃,extracted with 3×50 mL CH₂Cl₂, and dried over Na₂SO₄. Filtration,concentration of the filtrate, and drying gave a crude oil or solid.Further purification on a silica gel column (2-4% MeOH/CHCl₃, 0.2%NH₄OH) afforded the final product.

Preparation 3

100 mg of the above compound of formula 13 was dissolved in MeOH/H₂O(8:1). Sodium azide (7 equiv) and ammonium chloride (5.5 equiv) wereadded and the solution was heated to 60° C. for two days. The reactionwas worked up by quenching with 50 mL saturated NaHCO₃, extracted with3×50 mL CH₂Cl₂, and dried over Na₂SO₄. Filtration, concentration of thefiltrate, and drying gave a crude oil or solid. Further purification ona silica gel column (2% MeOH/CHCl₃, 0.2% NH₄OH) afforded the finalproduct.

Preparation 4

150-250 mg of the above compound of formula 13 was dissolved in 1-2 mLMeOH/H₂O or MeOH. To this was added the heteroaromatic reagentcorresponding to the R substituent specified in Table 1 (10-50 equiv)and a catalytic amount (20 mg) of pyridinium hydrochloride. The reactionmixture was heated at 45-50° C. for one to three days. The reaction wasthen quenched with 100 mL saturated NaHCO₃, extracted with 3×25 mLCH₂Cl₂, dried over Na₂SO₄, filtered, and concentrated to a solid. Thesolid was re-dissolved in 100 mL EtOAc and washed with 3×25 mL 2N NaOHto remove the excess reagent. Further purification on a silica gelcolumn (2-5% MeOH/CHCl₃, 0.2% NH₄OH) afforded the final product.

Preparation 5

50 mg of the above compound of formula 13 was dissolved in 1 mL of anamine corresponding to the R substituent specified in Table 1. A smallscoop of neutral alumina was added and the mixture was stirred at roomtemperature for seven days. The reaction was worked up by filteringthrough Celite™ (diatomaceous earth) and concentrated to a crude solid.Further purification on a silica gel column (5% MeOH/CHCl₃, 0.2% NH₄OH)afforded the final product.

Preparation 6

270 mg of the above compound of formula 13 was dissolved in 4 mLbenzene. To this was added excess K₂CO₃ and 0.5 mL of thiol. The mixturestirred at room temperature for 16 hours. The reaction was quenched with100 mL saturated NaHCO₃, extracted with 3×25 mL CH₂Cl₂, dried overNa₂SO₄, filtered, and concentrated to a solid. Further purification on asilica gel column (2% MeOH/CHCl₃, 0.2% NH₄OH) afforded the finalproduct.

Preparation 7

250 mg of the above compound of formula 13 was dissolved in 0.5 mLbis(2-hydroxyethyl)amine and 2 mL 2-propanol in a sealed tube. Acatalytic amount (20 mg) of pyridinium hydrochloride was added and thesolution was heated to 75° C. for seven days. The reaction was worked upby quenching with 50 mL saturated NaHCO₃, extracted with 3×50 mL CH₂Cl₂and dried over Na₂SO₄. Filtration, concentration of the filtrate, anddrying gave a crude oil or solid. Further purification on a silica gelcolumn (2% MeOH/CHCl₃, 0.2% NH₄H) afforded the final product.

Examples 33-68 below describe the preparation of compounds having thegeneral structure of formula 9 below wherein R is as defined in theexamples.

EXAMPLE 33

To a solution of the compound of formula 4 wherein R⁴ is H (0.059 g,0.08 mmol) in THF (2 mL) at 0° C. was added allylmagnesium bromide inEt₂O (1.0 M, 0.5 mL). After 2 hours at 0° C., stirring was continued atroom temperature for 12 hours. The reaction was diluted with a saturatedaqueous solution of sodium bicarbonate (10 mL) and EtOAc (20 mL). Afterseparation, the aqueous layer was washed with EtOAc (2×15 mL). Thecombined organic extracts were washed with a saturated aqueous solutionof sodium bicarbonate (20 mL) and brine (25 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.011 g (18% yield) ofthe compound of formula 9 wherein R is allyl: MS: 776 (TS).

EXAMPLE 34

To a solution of the compound of formula 4 wherein R⁴ is H (0.059 g,0.08 mmol) in DME (3 mL) at 0° C. was added vinylmagnesium bromide inTHF (1.0 M, 0.56 mL). After stirring at 0° C. for 1 hour and at roomtemperature for 1 hour, the reaction mixture was diluted with asaturated aqueous solution of sodium bicarbonate (10 mL) and EtOAc (10mL). After separation, the aqueous layer was washed with EtOAc (3×10mL). The combined organic extracts were washed with a saturated aqueoussolution of sodium bicarbonate (15 mL) and brine (20 mL), dried overNa₂SO₄ and concentrated under vacuum. Silica gel chromatography with.MeOH:CH₂Cl₂:NH₄OH (6:93:1) afforded 0.016 g (26% yield) of the compoundof formula 9 wherein R is vinyl: MS: 762 (FAB).

EXAMPLE 35

To a flask containing MgCl₂ (0.095 g, 1 mmol) and DME (1 mL) at 0° C.was added 2-thienyl lithium (1.0 M, 1.0 mL). After 0.5 hour, a solutionof the compound of formula 4 wherein R⁴ is H (0.073 g, 0.1 mmol) in DME(2 mL) was introduced and stirring was continued at 0° C. for 1 hour,then at room temperature for 0.5 hour. The reaction mixture was dilutedwith a saturated aqueous solution of sodium bicarbonate (10 mL) andEtOAc (15 mL). After separation, the aqueous layer was washed with EtOAc(3×10 mL). The combined organic extracts were washed with a saturatedaqueous solution of sodium bicarbonate (15 mL) and brine (20 mL), driedover Na₂SO₄ and concentrated under vacuum. Silica gel chromatographywith MeOH:CH₂Cl₂:NH₄OH (6:93:1) afforded 0.012 g (15% yield) of thecompound of formula 9 wherein R is 2-thienyl: MS: 817 (TS).

EXAMPLE 36

To a solution of the compound of formula 4 wherein R⁴ is H (0.147 g, 0.2mmol) in DME (10 mL) at 0° C. was added ethynylmagnesium bromide in THF(0.5 M, 2.8 mL). After stirring at 0° C. for 1 hour and at roomtemperature for 1 hour, the reaction mixture was diluted with water (20mL) and EtOAc (35 mL). After separation, the aqueous layer was washedwith EtOAc (3×25 mL). The combined organic extracts were washed with asaturated aqueous solution of sodium bicarbonate (30 mL) and brine (30mL), dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.068g (45% yield) of the compound of formula 9 wherein R is ethynyl: MS: 759(API).

EXAMPLE 37

To a solution of the compound of formula 4 wherein R⁴ is H (0.220 g, 0.3mmol) in DME (15 mL) at 0° C. was added 1-methyl-1-propenylmagnesiumbromide in THF (0.5 M, 4.2 mL). After stirring at room temperature for 3hours, the reaction mixture was diluted with a saturated aqueoussolution of sodium bicarbonate (20 mL) and EtOAc (30 mL). Afterseparation, the aqueous layer was washed with EtOAc (3×10 mL). Thecombined organic extracts were washed with a saturated aqueous solutionof sodium bicarbonate (25 mL) and brine (30 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.068 g (26% yield) ofthe compound of formula 9 wherein R is 1-methyl-1-propenyl: MS: 790(API).

EXAMPLE 38

To a solution of butylmagnesium bromide in THF (2.0 M, 1.0 mL) at 0° C.was added a solution of methyl propargyl ether (0.154 g, 0.2 mmol) inDME (3 mL). After stirring at 0° C. for 0.5 hour, a solution of thecompound of formula 4 wherein R⁴ is H (0.147 g, 0.2 mmol) in DME (7 mL)was added. After stirring at 0° C. for 0.5 hour and room temperature for4 hours, the reaction mixture was diluted with water (20 mL) and EtOAc(25 mL). After separation, the aqueous layer was washed with EtOAc (3×20mL). The combined organic extracts were washed with a saturated aqueoussolution of sodium bicarbonate (20 mL) and brine (25 mL), dried overNa₂SO₄ and concentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.081 g (50% yield) ofthe compound of formula 9 wherein R is 3-methoxy-1-propynyl: MS: 803(API).

EXAMPLE 39

To a solution of methylmagnesium bromide in Et₂O (3.0 M, 1.8 mL) at 0°C. was added a solution of 1-dimethylamino-2-propyne (0.154 g, 0.2 mmol)in THF (5 mL). After stirring at 0° C. for 6 hours, a solution of thecompound of formula 4 wherein R¹³ is H (0.147 g, 0.2 mmol) in DME (10mL) was added at room temperature. After stirring at room temperaturefor 3 hours, the reaction mixture was diluted with water (40 mL) andEtOAc (50 mL). After separation, the aqueous layer was washed with EtOAc(3×50 mL). The combined organic extracts were washed with a saturatedaqueous solution of sodium bicarbonate (40 mL) and brine (50 mL), driedover Na₂SO₄ and concentrated under vacuum. Silica gel chromatographywith MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 8:91:1) afforded 0.140 g (57% yield)of the compound of formula 9 wherein R is 3-dimethylamino-1-propyny MS:817 (API).

EXAMPLE 40

To a solution of methylmagnesium bromide in Et₂O (3.0 M, 1.8 mL) and DME(1 mL) at 0° C. was added a solution of 2-ethynylpyridine (0.186 g, 1.8mmol) in DME (2 mL). After stirring at 0° C. for 1 hour and roomtemperature for 1 hour, a solution of the compound of formula 4 whereinR⁴ is H (0.110 g, 0.15 mmol) in DME (7 mL) was added at roomtemperature. After stirring at room temperature for 3 hours, thereaction mixture was diluted with water (20 mL) and EtOAc (40 mL). Afterseparation, the aqueous layer was washed with EtOAc (3×30 mL). Thecombined organic extracts were washed with a saturated aqueous solutionof sodium bicarbonate (50 mL) and brine (50 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.066 g (53% yield) ofthe compound of formula 9 wherein R is 2-pyridylethynyl: MS: 836 (API).

EXAMPLE 41

To a round bottomed flask containing MgBr₂ (0.552 g, 3.0 mmol) andpropynyl lithium (0.069 g, 1.5 mmol) at 0° C. was added THF (5 mL).After 4 hours, a solution of the compound of formula 4 wherein R⁴ is H(0.110 g, 0.15 mmol) in DME (10 mL) was introduced at room temperatureand stirring was continued for 3 hours. The reaction mixture was dilutedwith water (30 mL) and EtOAc (30 mL). After separation, the aqueouslayer was washed with EtOAc (3×40 mL). The combined organic extractswere washed with a saturated aqueous solution of sodium bicarbonate (50mL) and brine (50 mL), dried over Na₂SO₄ and concentrated under vacuum.Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 7:92:1)afforded 0.060 g (52% yield) of the compound of formula 9 wherein R is1-propynyl: MS: 817 (TS).

EXAMPLE 42

To a solution of methylmagnesium bromide in Et₂O (3.0 M, 0.6 mL) mL) at0° C. was added a solution of propargyl alcohol (0.346 mL, 0.289 g, 2.25mmol) in THF (5 mL). After stirring at 0° C. for 3 hours, a solution ofthe compound of formula 4 wherein R⁴ is H (0.110 g, 0.15 mmol) in DME(10 mL) was added at room temperature. After stirring at roomtemperature for 2 hours, the reaction mixture was diluted with water (35mL) and EtOAc (50 mL). After separation, the aqueous layer was washedwith EtOAc (3×40 mL). The combined organic extracts were washed with asaturated aqueous solution of sodium bicarbonate (50 mL) and brine (50mL), dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 15:84:1) afforded 0.038g (32% yield) of the compound of formula 9 wherein R is3-hydroxy-1-propynyl: MS: 790 (API).

EXAMPLE 43

Palladium catalyst (20 mg, 10% Pd/C) was added to a solution of thecompound from example 42 in isopropanol (8 mL). The reaction vessel wasflushed and filled with hydrogen (50 psi) and shaken at room temperaturefor 24 hours. Filtration of an aliquot of the reaction mixture throughCelite™ and concentration under vacuum afforded the compound of formula9 wherein R is 3-hydroxy-1-propenyl: MS: 791 (API).

EXAMPLE 44

Palladium catalyst (20 mg, 10% Pd/C) was added to the remaining solutionfrom example 43 and the reaction vessel was flushed and filled withhydrogen (50 psi) and shaken at room temperature for 48 hours. Thereaction mixture was filtered through Celite™ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to8:91:1) afforded 0.018 g (57% yield) of the compound of formula 9wherein R is 3hydroxypropyl: MS: 793 (API)

EXAMPLE 45

Palladium catalyst (15 mg, 10% Pd/C) was added to a solution of thetitle compound from example 38 in isopropanol (8 mL). The reactionvessel was flushed and filled with hydrogen (50 psi) and shaken at roomtemperature for 24 hours. Filtration of an aliquot of the reactionmixture through Celite™ and concentration under vacuum afforded thecompound of formula 9 wherein R is 3-methoxy-1-propenyl: MS: 806 (API).

EXAMPLE 46

Palladium catalyst (15 mg, 10% Pd/C) was added to the remaining solutionfrom example 45 and the reaction vessel was flushed and filled withhydrogen (50 psi) and shaken at room temperature for 48 hours. Thereaction mixture was filtered through Celite™ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to7:92:1) afforded 0.017 g (73% yield) of the compound of formula 9wherein R is 3-methoxy-propyl: MS: 808 (API)

EXAMPLE 47

To a solution of the compound of formula 4 wherein R⁴ isbenzyloxycarbonyl (0.520 g, 0.6 mmol) in DME (6 mL) and TMEDA (2 mL) at−40° C. was added propynyl lithium (0.414 g, 9.0 mmol). After stirringat −40° C. for 2.5 hours, the reaction mixture was diluted with asaturated aqueous solution of ammonium chloride (30 mL) and EtOAc (30mL). After separation, the aqueous layer was washed with EtOAc (3×10mL). The combined organic extracts were washed with a saturated aqueoussolution of sodium bicarbonate (25 mL) and brine (30 mL), dried overNa₂SO₄ and concentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (4:95.6:0.4 to 6:93.6:0.4) afforded 0.157 g (29%yield) of the faster eluting diastereomer, along with 0.071 g (13%yield) of the slower eluting diastereomer and 0.070 g (13% yield) of amixture of the diastereomers.

A solution of the faster eluting diastereomer (0.157 g, 0.17 mmol) inMeOH (5 mL) was allowed to stir at 30° C. for 6 days. Upon concentrationunder vacuum, silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH(4:95.6:0.4 to 6:93.6:0.4) afforded 0.102 g (78% yield) of the compoundof formula 9 wherein R is 1-propynyl according to the followingconfiguration at the C-4″ carbon (MS: 774

A solution of the slower eluting diastereomer (0.071 g, 0.078 mmol) inMeOH (3 mL) was allowed to stir at 30° C. for 6 days. Upon concentrationunder vacuum, silica gel chromatography with MeOH:CH₂Cl₂:NH₄H(4:95.6:0.4 to 6:93.6:0.4) afforded 0.041 g (68% yield) of materialidentical to that described by the compound of Example 41 whichcorresponds to the compound of formula 9 wherein R is 1-propynylaccording to the following configuration at the C-4″ carbon (MS: 774(API)):

EXAMPLE 48

To a suspension of trimethylsulfonium tetrafluoroborate (1.03 g, 6.3mmol) in THF (40 mL) at −10° C. was added KHMDS (1.20 g, 6.0 mmol).After stirring below 0° C. for 0.5 hour, the reaction vessel was cooledto −78° C. and a solution of the compound of formula 4 wherein R¹³ isbenzyloxycarbonyl (2.60 g, 3 mmol) in DME (10 mL) was added. After 0.5hour, the reaction mixture was diluted with a saturated aqueous solutionof ammonium chloride (40 mL) and EtOAc (50 mL). After separation, theaqueous layer was washed with EtOAc (3×30 mL). The combined organicextracts were washed with brine (40 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (2:97.6:0.4 to 4:95.5:0.4) afforded 0.834 g (32%yield) of the compound of formula 5 wherein R⁴ is benzyloxycarbonyl (MS:881 (API)).

EXAMPLE 49

A solution of the compound of Example 48 (0.176 g, 0.2 mmol) in MeOH (5mL) was allowed to stir at 50° C. for 4 days. Upon concentration, silicagel chromatography with MeOH:CH₂Cl₂:NH₄OH (4:95.6:0.4 to 6:93.5:0.4)afforded 0.107 g (72% yield) of the compound of formula 5 wherein R⁴ ishydrogen and the epoxide moiety at C-4″ has the following configuration(MS: 748 (API)):

EXAMPLE 50

A solution of the compound of Example 48 (0.176 g, 0.2 mmol), potassiumiodide (2.32 g, 14 mmol) and cyclopropylamine (2.43 mL, 2.00 g, 35 mmol)in MeOH (30 mL) was allowed to stir at 50° C. for 2 days. Uponconcentration, the residue was dissolved in water (50 mL) and EtOAc (100mL). After separation, the aqueous layer was washed with EtOAc (3×50mL). The combined organic extracts were washed with a saturated aqueoussolution of sodium bicarbonate (50 mL) and brine (40 mL), dried overNa₂SO₄ and concentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (4:95.6:0.4 to 6:93.5:0.4) afforded 0.377 g (69%yield) of the compound of formula 9 wherein R is cyclopropylaminomethylaccording to the following configuration at the C-4″ carbon (MS: 805(API)):

EXAMPLE 51

A solution of the compound of Example 48 (0.176 g, 0.2 mmol),tetrabutylammonium iodide (0.739 g, 2.0 mmol) and butylamine (0.395 mL,0.293 g, 4 mmol) in MeOH (5 mL) was allowed to stir at 50° C. for 2days. Upon concentration, the residue was dissolved in water (20 mL) andEtOAc (20 mL). After separation, the aqueous layer was washed with EtOAc(3×20 mL). The combined organic extracts were washed with brine (40 mL),dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (4:95.6:0.4 to 6:93.5:0.4)afforded 0.088 g (54% yield) of the compound of formula 9 wherein R ispropylaminomethyl according to the following configuration at the C-4″carbon (MS: 821 (API)):

EXAMPLE 52

To a solution of a compound of formula 4 wherein R⁴ is benzyloxycarbonyland the hydrogen attached to the C-9a nitrogen is replaced bybenzyloxycarbonyl (0.500 g, 0.499 mmol) in THF (15 mL) 0° C. was addedmethylmagnesium bromide in Et₂O (3.0 M, 1.2 mL). After 20 minutes, thereaction was diluted with EtOAc (30 mL) and water (50 mL). Afterseparation, the aqueous layer was washed with EtOAc (3×35 mL). Thecombined organic extracts were washed with a 10% aqueous solution ofsodium bicarbonate (100 mL) and brine (120 mL), dried over Na₂SO₄ andconcentrated under vacuum to afford 0.500 g (98% yield) of an off-whitefoam. (MS: 1017, 845 (API)).

Palladium catalyst (0.250 g, 10% Pd/C) was added to a solution of thecompound described above (0.500 g 0.491 mmol) in isopropanol (50 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 48 hours. Additional palladium catalyst(0.250 g,10% Pd/C) was added and hydrogenation was continued at 50 psifor 24 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. The resulting oil was dissolved inisopropanol (50 mL), palladium catalyst was added (0.312 g, 10% Pd/C),and hydrogenation was continued at 50 psi for 24hours. Additionalpalladium catalyst (0.170 g, 10% Pd/C) was added and hydrogenation wascontinued at 50 psi for 24 hours. The reaction mixture was filteredthrough Celite™ and concentrated under vacuum. Silica gel chromatographywith MeOH:CH₂Cl₂:NH₄OH (8:91:1 to 10:89:1) afforded 0.120 g (33% yield)of the compound of formula 9 wherein R is methyl according to thefollowing configuration at the C-4″ carbon (MS: 749 (API)):

EXAMPLE 53

To a solution of a compound of formula 4 wherein R⁴ is benzyloxycarbonyland the hydrogen attached to the C-9a nitrogen is replaced bybenzyloxycarbonyl (0.101 g, 0.101 mmol) in THF (2 mL) at −78° C. wasadded phenylmagnesium bromide in THF (1.01 M, 1.0 mL). After 15 minutes,stirring was continued 0° C. for 1 hour, then at room temperature for 12hours. The reaction was diluted with a 10% aqueous solution of sodiumbicarbonate (10 mL) and EtOAc (20 mL). After separation, the aqueouslayer was washed with EtOAc (3×15 mL). The combined organic extractswere washed with a 10% aqueous solution of sodium bicarbonate (20 mL)and brine (25 mL), dried over Na₂SO₄ and concentrated under vacuum.Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (5:94:1 to 25:74:1)afforded 0.048 g (45% yield) of a white foam (MS: 1080 (LSIMS)).

Palladium catalyst (0.024 g, 10% Pd/C) was added to a solution of thecompound described above (0.024 g, 0.022 mmol) in methanol (15 mL). Thereaction vessel was flushed and filled with hydrogen (50 psi) and shakenat room temperature for 24 hours. The reaction mixture was filteredthrough Celite™ and concentrated under vacuum. Silica gel chromatographywith MeOH:CH₂Cl₂:NH₄OH (5:94.5:1 to 10:89:1) afforded 0.010 g (28%yield) of the compound of formula 9 wherein R is phenyl: MS: 811(LSIMS).

EXAMPLE 54

To a solution of the starting compound used in Example 53 (0.300 g, 0.30mmol) in THF (3 mL) at 0° C. was added n-butylmagnesium chloride in THF(2.0 M, 1.5 mL). After 20 minutes the reaction was diluted with waterand EtOAc (20 mL). After separation, the aqueous layer was washed withEtOAc (3×50 mL). The combined organic extracts were washed with a 10%aqueous solution of sodium bicarbonate (50 mL) and brine (55 mL), driedover Na₂SO₄ and concentrated under vacuum to afford 0.295 g (93% yield)of an off-white foam (MS: 1060 (FAB)).

Palladium catalyst (0.087 g, 10% Pd/C) was added to a solution of thecompound described above (0.087 g, 0.082 mmol) in isopropanol (15 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 24 hours. Additional palladium catalyst(0.087 g, 10% Pd/C) was added and hydrogenation was continued at 50 psifor 60 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (5:94.5:0.5 to 10:89:1) afforded 0.010 g (28% yield)of the compound of formula 9 wherein R is n-butyl: MS: 792 (API).

EXAMPLE 55

To a solution of the starting compound used in Example 53 (0.200 g, 0.20mmol) in THF (2 mL) at 0° C. was added ethylmagnesium bromide in THF(1.0 M, 2.0 mL). After 20 minutes the reaction was diluted with waterand EtOAc (20 mL). After separation, the aqueous layer was washed withEtOAc (3×30 mL). The combined organic extracts were washed with a 10%aqueous solution of sodium bicarbonate (50 mL) and brine (55 mL), driedover Na₂SO₄ and concentrated under vacuum. Silica gel chromatographywith MeOH:CH₂Cl₂:NH₄OH (5:94.5:0.5 to 20:79:1) afforded 0.079 g (38%yield) of a white foam (MS: 1033 (LSIMS)).

Palladium catalyst (0.035 g, 10% Pd/C) was added to a solution of thecompound described above (0.079 g, 0.077 mmol) in ethanol (20 mL). Thereaction vessel was flushed and filled with hydrogen (50 psi) and shakenat room temperature for 24 hours. Additional palladium catalyst (0.036g, 10% Pd/C) was added and hydrogenation was continued at 50 psi for 24hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum, affording 0.056 g (96% yield) of the compoundof formula 9 wherein R is ethyl: MS: 763 (TS).

EXAMPLE 56

To a solution of the starting compound used in Example 53 (0.300 g, 0.30mmol) in THF (3 mL) at 0° C. was added isopropenylmagnesium chloride inTHF (0.5 M, 6.0 mL). After 20 minutes the reaction was diluted withwater and EtOAc (20 mL). After separation, the aqueous layer was washedwith EtOAc (3×30 mL). The combined organic extracts were washed with a10% aqueous solution of sodium bicarbonate (50 mL) and brine (55 mL),dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (3:96.9:0.1 to 20:79.9:0.1)afforded 0.063 g (20% yield) of a white foam (MS: 1045 (LSIMS)).

Palladium catalyst (0.075 g, 10% Pd/C) was added to a solution of thecompound described above (0.150 g, 0.165 mmol) in ethanol (30 mL). Thereaction vessel was flushed and filled with hydrogen (50 psi) and shakenat room temperature for 24 hours. Additional palladium catalyst (0.075g, 10% Pd/C) was added and hydrogenation was continued at 50 psi for 24hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.024 g (19% yield) ofthe compound of formula 9 wherein R is isopropenyl: MS: 775 (TS).

EXAMPLE 57

To a solution of the starting compound used in Example 53 (0.750 g, 0.75mmol) in THF (12 mL) at 0° C. was added allylmagnesium chloride in THF(2.0 M, 3.0 mL). After 15 minutes the reaction was diluted with waterand EtOAc (40 mL). After separation, the aqueous layer was washed withEtOAc (3×50 mL). The combined organic extracts were washed with a 10%aqueous solution of sodium bicarbonate (100 mL) and brine (100 mL),dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 15:84:1) afforded 0.530g (68% yield) of an off-white foam (MS: 1044, 910 (API)).

Palladium catalyst (0.175 g, 10% Pd/C) was added to a solution of thecompound described above (0.350 g, 0.335 mmol) in isopropanol (100 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 24 hours. Additional palladium catalyst(0.150 g, 10% Pd/C) was added and hydrogenation was continued at 50 psifor 24 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.148 g (57% yield) ofthe compound of formula 9 wherein R is propyl: MS: 778 (API).

EXAMPLE 58

To a solution of the compound used as a starting material in Example 53(0.750 g. 0.75 mmol) in THF (12 mL) at 0° C. was added allylmagnesiumchloride in THF (2.0 M, 3.0 mL). After 15 minutes the reaction wasdiluted with water and EtOAc (40 mL). After separation, the aqueouslayer was washed with EtOAc (3×50 mL). The combined organic extractswere washed with a 10% aqueous solution of sodium bicarbonate (100 mL)and brine (100 mL), dried over Na₂SO₄ and concentrated under vacuum.Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 15:84:1)afforded 0.530 g (68% yield) of an off-white foam (MS: 1044 (API)).

A solution of the compound described above (0.104 g, 0.100 mmol) and(1S)-(+)-10-camphor sulfonic acid (0.046 g, 0.200 mmol) in MeOH (4 mL)was cooled to −78° C. and treated with ozone until a deep blue colorpersisted. The reaction was purged with oxygen, dimethylsulfide (0.13mL, 1.76 mmol) and pyridine (0.20 mL, 2.42 mmol) were added and stirringwas continued for 12 hours. CH₂Cl₂ (30 mL) and 10% aqueous solution ofsodium bicarbonate (10 mL) were added, the layers were separated and theaqueous layer was extracted with CH₂Cl₂ (3×30 mL). The combined organicextracts were washed with a 10% aqueous solution of sodium bicarbonate(50 mL) and brine (50 mL), dried over Na₂SO₄ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to10:89:1) afforded 0.024 g (23% yield) of an off-white foam (MS: 912(API)).

To a solution of the compound described above (0.022 g, 0.024 mmol) inMeOH (1 mL) was added sodium borohydride (0.001 g, 0.024 mmol).Additional sodium borohydride (0.004 g, 1.00 mmol) was added over aperiod of 3 hours. The reaction mixture was diluted with CH₂Cl₂ (30 mL)and 10% sodium bicarbonate solution (20 mL). After separation, theaqueous layer was extracted with CH₂Cl₂ (3×30 mL). The combined organicextracts were washed with a 10% aqueous solution of sodium bicarbonate(50 mL) and brine (50 mL), dried over Na₂SO₄ and concentrated undervacuum to afford 0.022 g (100% yield) of a yellow foam (MS: 914 (API)).

Palladium catalyst (0.012 g. 10% Pd/C) was added to a solution of thecompound described above (0.022 g, 0.024 mmol) in isopropanol (10 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 24 hours. Additional palladium catalyst(0.020 g. 10% Pd/C) was added and hydrogenation was continued at 50 psifor 24 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂C₆:NH₄OH (8:91:1 to 10:89:1) afforded 0.005 mg (23% yield) ofthe compound of formula 9 wherein R is 2-hydroxyethyl: MS: 779 (API).

EXAMPLE 59

To a solution of the starting compound used in Example 53 (0.750 g, 0.75mmol) in THF (12 mL) at 0° C. was added allylmagnesium chloride in THF(2.0 M, 3.0 mL). After 15 minutes the reaction was diluted with waterand EtOAc (40 mL). After separation, the aqueous layer was washed withEtOAc (3×50 mL). The combined organic extracts were washed with a 10%aqueous solution of sodium bicarbonate (100 mL) and brine (100 mL),dried over Na₂SO₄ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 15:84:1) afforded 0.530g (68% yield) of an off-white foam (MS: 1044 (API)).

A solution of the compound described above (0.104 g, 0.100 mmol) and(1S)-(+)-10-camphor sulfonic acid (0.046 g, 0.200 mmol) in MeOH (4 mL)was cooled to −78° C. and treated with ozone until a deep blue colorpersisted. The reaction was purged with oxygen, dimethylsulfide (0.13mL, 1.76 mmol) and pyridine (0.20 mL, 2.42 mmol) were added and stirringwas continued for 12 hours. CH₂Cl₂ (30 mL) and 10% aqueous solution ofsodium bicarbonate (10 mL) were added, the layers were separated and theaqueous layer was extracted with CH₂Cl₂ (3×30 mL). The combined organicextracts were washed with a 10% aqueous solution of sodium bicarbonate(50 mL) and brine (50 mL), dried over Na₂SO₄ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to10:89:1) afforded 0.024 g (23% yield) of an off-white foam (MS: 912(API)).

Palladium catalyst (0.040 g, 10% Pd/C) was added to a solution of thecompound described above (0.057 g, 0.063 mmol) in isopropanol (15 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 24 hours. Additional palladium catalyst(0.040 g. 10% Pd/C) was added and hydrogenation was continued at 50 psifor 24 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.010 g (15% yield) ofthe compound of formula 9 wherein R is formylmethyl: MS: 777 (API).

EXAMPLE 60

To a solution of 2-bromopyridine (0.474 g, 3.0 mmol) in THF (5 mL) at−78° C. was added n-butyl lithium (3.0 M, 1.2 mL) at −78° C. After 40minutes, the solution was transferred via a cannula cooled with a dryice jacket to a flask containing MgCl₂ (0.428 g, 4.5 mmol) and ether (4mL) at −78° C. After 15 minutes, a solution of a compound of formula 4wherein R⁴ is benzyloxycarbonyl (0.260 g, 0.3 mmol) in THF (3 mL) at−78° C. was introduced and stirring was continued allowing the reactionto warm to room temparature over several hours. After 3.5 hours, thereaction mixture was diluted with a saturated aqueous solution of sodiumbicarbonate (20 mL) and EtOAc (30 mL). After separation, the aqueouslayer was washed with EtOAc (3×50 mL). The combined organic extractswere washed with a saturated aqueous solution of sodium bicarbonate (50mL) and brine (60 mL), dried over Na₂SO₄ and concentrated under vacuum.Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93.3:0.7 to 10:89:1)afforded 0.023 g (9.5% yield) of the compound of formula 9 wherein R is2-pyridyl: MS: 812 (API).

EXAMPLE 61

To a round bottom flask containing n-butyl lithium (3.0 M, 1.62 mL) indiethyl ether (15 mL) at −78° C. was added chilled (−78° C.)3-bromopyridine (0.790 g, 5 mmol) via a cannula cooled with a dry icejacket. Stirring continued at −78° C. for 35 minutes. A suspension ofMgBr₂ diethyl ethereate (0.114 g, 0.440 mmol) in diethyl ether (3 mL) at−78° C. was added via a cannula cooled with a dry ice jacket to the3-pyridyl lithium solution. A solution of a compound of formula 4wherein R⁴ is benzyloxycarbonyl (0.347 g, 0.400 mmol) in diethyl ether(3 mL) at −78° C. was introduced via cannula. Stirring continued at −78°C. for 2 hours and slowly allowed to warm to 0° C. over 3 hours. Thereaction mixture was diluted with a saturated aqueous solution of sodiumbicarbonate (20 mL) and EtOAc (30 mL). After separation, the aqueouslayer was washed with EtOAc (3×50 mL). The combined organic extractswere washed with a saturated aqueous solution of sodium bicarbonate (50mL) and brine (60 mL), dried over Na₂SO₄ and concentrated under vacuum.Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (4:95.4:0.6 to 20:79:1)afforded 0.075 g (26% yield) of a white foam (MS: 947, 812 (API)).

Palladium catalyst (0.073 g, 10% Pd/C) was added to a solution of thecompound described above (0.073 g, 0.077 mmol) in isopropanol (30 mL).The reaction vessel was flushed and filled with hydrogen (50 psi) andshaken at room temperature for 48 hours. The reaction mixture wasfiltered through Celite™ and concentrated under vacuum. Silica gelchromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to 8:91:1) afforded 0.032g (51% yield) of the compound of formula 9 wherein R is 3-pyridyl: MS:812 (API).

EXAMPLE 62

To a solution of methyl magnesium bromide in diethyl ether (3.0 M, 1.8mL) at 0° C. was added a solution of 5-hexynenitrile (0.63 mL, 6.00mmol) in THF (5 mL). After stirring at 0° C. for 6 hours, a solution ofthe compound of formula 4 wherein R⁴ is H (0.220 g, 0.300 mmol) in DME(10 mL) was added and stirring was continued at 0° C. for 0.5 hour, thenat room temperature for 4 hours. The reaction mixture was diluted withwater (20 mL) and EtOAc (25 mL), the layers were separated and theaqueous layer was washed with EtOAc (3×20 mL). The combined organicextracts were washed with a saturated aqueous solution of sodiumbicarbonate (20 mL) and brine (25 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (6:93:1 to 10:89:1) afforded 0.035 g (14% yield) ofthe compound of formula 9 wherein R is 6-cyano-1-pentynyl: MS: 827(API).

EXAMPLE 63

To a solution of the compound of Example 49, except wherein R⁴ isbenzyloxycarbonyl, (0.101 g, 0.115) in DME (3 mL) was added LiAlH₄ (1.0M, 2.1 mL) dropwise. After 10 minutes the reaction mixture was treatedsequentially with water (0.044 mL), 15% NaOH solution (0.044 mL), andwater (0.132 mL), then stirred at rt for 0.5 hour. The mixture wasdiluted with EtOAc (20 mL) and water (20 mL). After separation theaqueous layer was extracted with EtOAc (3×30 mL). The combined organicextracts were washed with a saturated aqueous solution of sodiumbicarbonate (50 mL) and brine (60 mL), dried over Na₂SO₄ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (3:96.5:0.5 to 3.5:95:0.5) afforded 0.042 g (49%yield) of the compound of formula 9 wherein R is methyl according to thefollowing configuration at the C-4″ carbon (MS: 749 (API)):

EXAMPLE 64

To a solution of 1-methylimidazole (0.41 g, 4.99 mmol) in THF (5 ml) at−78° C. was added n-butyl lithium (2.5M,2.02 ml). After 45 minutes at−78° C. the solution was added via cannula to a flask containing MgCl₂(0.71 g, 7.49 mmol) and THF (5 mL) at 0° C. After 1.5 hours at 0° C., asolution of the starting compound used in Example 53 (0.500 g, 0.499mmol) in DME (2 mL) was introduced and stirring was continued at 0° C.for 1 hour. The reaction mixture was diluted with a saturated aqueoussolution of sodium bicarbonate (100 mL) and EtOAc (100 mL). Afterseparation, the aqueous layer was washed with EtOAc (3×100 mL). Thecombined organic extracts were washed with a saturated aqueous solutionof sodium bicarbonate (100 mL) and brine (100 mL), dried over Na₂SO₄ andconcentrated under vacuum to afford 0.660 g of a yellow foam (MS: 949(API)).

Palladium catalyst (0.700 g. 10% Pd/C) was added to a solution of thecompound described above in isopropanol (60 mL). The reaction vessel wasflushed and filled with hydrogen (50 psi) and shaken at room temperaturefor 24 hours. Additional palladium catalyst (0.500 g, 10% Pd/C) wasadded and hydrogenation was continued at 50 psi for 24 hours. Thereaction mixture was filtered through Celite™ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (1:98:1 to8:91:1) afforded 0.052 g (13% yield) of the compound of formula 9wherein R is 1-methylimidazol-2-yl: MS: 816 (API).

EXAMPLE 65

To a solution of furan (0.34 g, 4.99 mmol) in THF (5ml) at −78° C. wasadded n-butyl lithium (2.5M, 1.98 ml). After 0.5 hour at −78° C. thesolution was added to a flask containing MgCl₂ (0.71 g, 7.49 mmol) andTHF (5 mL) at 0° C. After 1.5 hours at 0° C., a solution of the startingcompound used in Example 53 (0.500 g, 0.499 mmol) in DME (2 mL) wasintroduced and stirring was continued at 0° C. for 1 hour, then at roomtemperature for 1 hour. The reaction mixture was diluted with asaturated aqueous solution of sodium bicarbonate (100 mL) and EtOAc (100mL). After separation, the aqueous layer was washed with EtOAc (3×100mL). The combined organic extracts were washed with a saturated aqueoussolution of sodium bicarbonate (100 mL) and brine (100 mL), dried overNa₂SO₄ and concentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (1:98:1 to 8:91:1) afforded 0.096 g (24% yield) of awhite foam (MS: 935 (API)).

Palladium catalyst (0.100 g, 10% Pd/C) was added to a solution of thecompound described above in isopropanol (15 mL). The reaction vessel wasflushed and filled with hydrogen (50 psi) and shaken at room temperaturefor 72 hours. The reaction mixture was filtered through Celite™ andconcentrated under vacuum. Silica gel chromatography withMeOH:CH₂Cl₂:NH₄OH (1:98:1 to 8:91:1) afforded 0.053 g (13% yield) of thecompound of formula 9 wherein R is 2-furyl: MS: 802 (API).

EXAMPLE 66

To a solution of N-methylpyrrole (0.184 g, 2.31 mmol) in THF (4 ml) at−78° C. was added n-butyl lithium (2.5M, 0.93 ml). The solution waswarmed to room temperature over 1 hour and then added via cannula to aflask containing MgCl₂ (0.329 g, 3.46 mmol) and Et₂O (4 mL) at roomtemperature. After 1 hour, a solution of the compound of formula 4wherein R⁴ is benzyloxycarbonyl (0.200 g, 0.231 mmol) in THF (2 mL) wasintroduced and stirring was continued at room temperature for 45minutes. The reaction mixture was diluted with a saturated aqueoussolution of sodium bicarbonate (50 mL) and EtOAc (50 mL). Afterseparation, the aqueous layer was washed with EtOAc (3×50 mL). Thecombined organic extracts were washed with a saturated aqueous solutionof sodium bicarbonate (50 mL) and brine (50 mL), dried over Na₂SO₄ andconcentrated under vacuum to afford 0.293 g of a yellow foam (MS: 949(API)).

Palladium catalyst (0.324 g, 10% Pd/C) was added to a solution of thecompound described above in isopropanol (30 mL). The reaction vessel wasflushed and filled with hydrogen (50 psi) and shaken at room temperaturefor 24 hours. Additional palladium catalyst (0.300 g, 10% Pd/C) wasadded and hydrogenation was continued at 50 psi for 24 hours. Thereaction mixture was filtered through Celite™ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (6:93:1 to8:91:1) afforded 0.033 g (18% yield) of the compound of formula 9wherein R is 1-methyl-2-pyrrolyl: MS: 814 (API).

EXAMPLE 67

To a solution of unpurified compound prepared as described in Example 39(0.480 g) in isopropanol (40 mL) was added platinum oxide (0.115 g,0.505 mmol). The reaction vessel was flushed and filled with hydrogen(50 psi) and shaken at room temperature for 24 hours. Filtration of analiquot of the reaction mixture through Celite™ and concentration undervacuum afforded the compound of formula 9 wherein R is3-dimethylamino-1-propenyl: MS: 819 (API).

EXAMPLE 68

Platinum oxide (0.076 g, 0.335 mmol) was added to the remaining solutionfrom Example 67 and the reaction vessel was flushed and filled withhydrogen (50 psi) and shaken at room temperature for 96 hours. Thereaction mixture was filtered through Celite™ and concentrated undervacuum. Silica gel chromatography with MeOH:CH₂Cl₂:NH₄OH (4:95:1 to6:93:1) afforded 0.069 g (15% yield) of the compound of formula 9wherein R is 3-dimethylpropyl: MS: 821 (API).

TABLE 2 The compounds of Examples 69-81 have the general structure offormula 10 below with the R substituents indicated in the table below.The compounds of Examples 69-82 were prepared following the proceduresof Examples 50 and 51, referred to above, with the reaction periodspecified in the table below. In the table, the yield and mass spectra(“Mass Spec”) data apply to the final product.

Reaction Time Example R (hours) Yield (%) Mass Spec  69 1-imidazolyl 7260 816  70 n-propylamino 48 55 807  71 dimethylamino 24 42 793  72methylamino 120 55 779  73 ethylamino 120 58 793  74 isopropylamino 4844 806  75 isobutylamino 48 27 821  76 trimethyleneimino 24 31 804  77allylamino 24 22 804  78 cyclopropylmethylamino 24 34 818  79N-ethylmethylamino 48 16 820  80 t-butylamino 96 30 821  81 diethylamino168 25 820  81(a)

48 75 818.5  81(b)

96 95 832.6  82 4-methoxybenzylamino 48 21.7 884.6  834-nitrobenzylamino 48 8 899.7  84 4-chlorobenzylamin 48 25.5 888.6  853,4-difluorobenzylamin 48 14.5 890.6  85 3-pyridylmethylamino 48 21.0855.6  86 4-trifluoromethylbenzyl- 48 16.5 922.6 amino  872,6-difluorobenzylamino 48 11.0 890.6  88 benzylamino 96 62 854.7  894-fluorobenzylamino 48 50.9 872.7  90 3-fluorobenzylamino 48 32.7 872.7 91 2-fluorobenzylamino 48 39.6 872.7  92 2,4-difluorobenzylamino 4824.6 890.1  93 2,5-difluorobenzylamino 48 28.1 890.1  943,5-difluorobenzylamino 48 35.6 890.1  95 1-(4- 48 44.7 927.6fluorophenyl)piperazine  96 2-trifluoromethylbenzyl- 48 32.7 922.5 amino 97 4-trifluoromethylbenzyl- 48 28.6 938.1 amino  983-trifluoromethylbenzyl- 48 262 922.6 amino  99 2-fluorophenylethyl- 4833.5 886.2 amino 100 3-fluorophenylethyl- 48 28.7 886.1 amino 1014-pyridylmethylamino 48 46 855.2 102 methyl,3- 72 28.8 869.6pyridylmethylamino 103 4-hydroxy-3- 48 12.0 900.1 methoxybenzylamino 104piperonylamino 48 14.0 898.1 105 3-methoxybenzylamino 48 33.0 884.1 1062-methoxybenzylamino 48 24.0 884.5 107 2-pyridylmethylamino 48 28.9855.1

What is claimed is:
 1. A pharmaceutical composition for the treatment ofa bacterial infection or a protozoal infection in a mammal, fish, orbird which comprises: (a) a therapeutically effective amount of acompound of the formula

or a pharmaceutically acceptable salt thereof, wherein, R³ is —CH₂NR⁸R¹⁵or —CH₂SR⁸; R⁴ is H, acetyl or benzyloxycarbonyl; each R⁶ and R⁷ isindependently H, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10 memberedheteroaryl), wherein m is an integer ranging from 0 to 4; each R⁸ isindependently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,—(CH₂)_(q)CR¹¹R¹²(CH₂)_(r)NR¹³R¹⁴ wherein q and r are each independentlyan integer ranging from 0 to 3 except q and r are not both 0,—(CN₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein the foregoingR⁸ groups, except H, are optionally substituted by 1 to 3 R¹⁶ groups; orwhere R⁸ is as —CH₂NR⁸R¹⁵, R¹⁵ and R⁸ may be taken together to form a4-10 membered monocyclic or polycyclic saturated ring or a 5-10 memberedheteroaryl ring, wherein said saturated and heteroaryl rings optionallyinclude 1 or 2 heteroatoms selected from the group consisting of O, Sand —N(R⁸)—, in addition to the nitrogen to which R¹⁵ and R⁸ areattached, said saturated ring optionally includes 1 or 2 carbon-carbondouble or triple bonds, and said saturated and heteroaryl rings areoptionally substituted by 1 to 3 R¹⁶ groups; each R⁹ and R¹⁰ isindependently H or C₁-C₆ alkyl; each R¹¹, R¹², R¹³ and R¹⁴ isindependently selected from the group consisting of H, C₁-C₁₀ alkyl,—(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein the foregoingR¹¹, R¹², R¹³ and R¹⁴ groups, except H, are optionally substituted by 1to 3 R¹⁶ groups; or R¹¹ and R¹³ are taken together to form —(CH₂)_(p)—wherein p is an integer ranging from 0 to 3 such that a 4-7 memberedsaturated ring is formed that optionally includes 1 or 2 carbon-carbondouble or triple bonds; or R¹³ and R¹⁴ are taken together to form a 4-10membered monocyclic or polycyclic saturated ring or a 5-10 memberedheteroaryl ring, wherein said saturated and heteroaryl rings optionallyinclude 1 or 2 heteroatoms selected from the group consisting of O, Sand —N(R⁸)—, in addition to the nitrogen to which R¹³ and R¹⁴ areattached, said saturated ring optionally includes 1 or 2 carbon-carbondouble or triple bonds, and said saturated and heteroaryl rings areoptionally substituted by 1 to 3 R¹⁶ groups; R¹⁵ is H, C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl, wherein the foregoing R¹⁵ groups areoptionally substituted by 1 to 3 substituents independently selectedfrom the group consisting of halo and —OR⁹; each R¹⁶ is independentlyselected from the group consisting of halo, cyano, nitro,trifluoromethyl, azido, —C(O)R¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷, —NR⁶C(O)R⁷,—C(O)NR⁶R⁷, —NR⁶R⁷, hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy,—(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein said aryl andheteroaryl substituents are optionally substituted by 1 or 2substituents independently selected from the group consisting of halo,dyano, nitro, trifluoromethyl, azido, —C(O)R¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷,—NR⁶C(O)R⁷, —C(O)NR⁶R₇, —NR⁶R⁷, hydroxy, C₁-C₆ alkyl, and C₁-C₆ alkyoxy;each R¹⁷ is independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl),and —(CH₂)_(m)(5-10 membered heteroaryl), wherein m is an integerranging from 0 to 4; with the proviso that R⁸ is not H where R is—CH²SR⁸; and (b) a pharmaceutically acceptable carrier.
 2. A method oftreating a bacterial infection or a protozoal disorder selected from abacterial infection, a protozoal infection in a mammal, fish, or birdwhich comprises administering to said mammal, fish or bird atherapeutically effective amount of the formula

or a pharmaceutically acceptable salt thereof, wherein, R³ is —CH₂NR⁸R¹⁵or —CH₂SR⁸; R⁴ is H, acetyl or benzyloxycarbonyl; each R⁶ and R⁷ isindependently H, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10 memberedheteroaryl), wherein m is an integer ranging from 0 to 4; each R⁸ isindependently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,—(CH₂)_(q)CR¹¹R¹² (CH₂)_(r)NR¹³R¹⁴ wherein q and r are eachindependently an integer ranging from 0 to 3 except q and r are not both0, —(CH₂)_(m)(C₆-C₁₀ aryl), or —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein the foregoingR⁸ groups, except H, are optionally substituted by 1 to 3 R¹⁶ groups; orwhere R⁸ is as —CH₂NR⁸R¹⁵, R¹⁵ and R⁸ may be taken together to form a4-10 membered monocyclic or polycyclic saturated ring or a 5-10 memberedheteroaryl ring, wherein said saturated and heteroaryl rings optionallyinclude 1 or 2 heteroatoms selected from the group consisting of O, Sand —N(R⁸)—, in addition to the nitrogen to which R¹⁵ and R⁸ areattached, said saturated ring optionally includes 1 or 2 carbon-carbondouble or triple bonds, and said saturated and heteroaryl rings areoptionally substituted by 1 to 3 R¹⁶ groups; each R⁹ and R¹⁰ isindependently H or C₁-C₆ alkyl; each R¹¹, R¹², R¹³ and R¹⁴ isindependently selected from the group consisting of H, C₁-C₁₀ alkyl,—(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein the foregoingR¹¹, R¹², R¹³ and R¹⁴ groups, except H, are optionally substituted by 1to 3 R¹⁶ groups; or R¹¹ and R¹³ are taken together to form —(CH₂)_(p)—wherein p is an integer ranging from 0 to 3 such that a 4-7 memberedsaturated ring is formed that optionally includes 1 or 2 carbon-carbondouble or triple bonds; or R¹³ and R¹⁴ are taken together to form a 4-10membered monocyclic or polycyclic saturated ring or a 5-10 memberedheteroaryl ring, wherein said saturated and heteroaryl rings optionallyinclude 1 or 2 heteroatoms selected from the group consisting of O, Sand —N(R⁸)—, in addition to the nitrogen to which R¹³ and R¹⁴ areattached, said saturated ring optionally includes 1 or 2 carbon-carbondouble or triple bonds, and said saturated and heteroaryl rings areoptionally substituted by 1 to 3 R¹⁶ groups; R¹⁵ is H, C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl, wherein the foregoing R¹⁵ groups areoptionally substituted by 1 to 3 substituents independently selectedfrom the group consisting of halo and —OR⁹; each R¹⁶ is independentlyselected from the group consisting of halo, cyano, nitro,trifluoromethyl, azido, —C(O)R¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷, —NR⁶C(O)R⁷,—C(O)NR⁶R⁷, —NR⁶R⁷, hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy,—(CH₂)_(m)(C₆-C₁₀ aryl), and —(CH₂)_(m)(5-10 membered heteroaryl),wherein m is an integer ranging from 0 to 4, and wherein said aryl andheteroaryl substituents are optionally substituted by 1 or 2substituents independently selected from the group consisting of halo,dyano, nitro, trifluoromethyl, azido, —C(O)R¹⁷, —C(O)OR¹⁷, —OC(O)OR¹⁷,—NR⁶C(O)R⁷, —C(O)NR⁶R₇, —NR⁶R⁷, hydroxy, C₁-C₆ alkyl, and C₁-C₆ alkyoxy;each R¹⁷ is independently selected from the group consisting of H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, —(CH₂)_(m)(C₆-C₁₀ aryl),and —(CH₂)_(m)(5-10 membered heteroaryl), wherein m is an integerranging from 0 to 4; with the proviso that R⁸ is not H where R is—CH²SR⁸.
 3. The pharmaceutical composition of claim 1 wherein R³ and—CH₂NR¹⁵R⁸ and R¹⁵ and R⁸ are independently selected from H, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, wherein the foregoing R¹⁵ andR⁸ groups, except H, are optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of hydroxy, halo andC₁-C₆-alkoxy.
 4. The pharmaceutical composition of claim 3 wherein R¹⁵and R⁸ are each independently selected from the group consisting of H,methyl, ethyl, allyl, n-butyl, isobutyl, 2-methoxyethyl, cyclopentyl,3-methoxypropyl, 3-ethoxypropyl, n-propyl, isopropyl, 2-hydroxyethyl,cyclopropyl, 2,2,2-trifluoroethyl, 2-propynyl, sec-butyl, tert-butyl,and n-hexyl.
 5. The pharmaceutical composition of claim 4 wherein R⁴ isH, R¹⁵ is H, R⁸ is n-propyl.
 6. The method of claim 2 wherein R³ is—CH₂NR¹⁵R⁸ and R¹⁵ and R⁸ are independently selected from H, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, wherein the foregoing R¹⁵ andR⁸ groups, except H, are optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of hydroxy, halo andC₁-C₆-alkoxy.
 7. The method of claim 2 wherein R¹⁵ and R⁸ are eachindependently selected from the group consisting of H, methyl, ethyl,allyl, n-butyl, isobutyl, 2-methoxyethyl, cyclopentyl, 3-methoxypropyl,3-ethoxypropyl, n-propyl, isopropyl, 2-hydroxyethyl, cyclopropyl,2,2,2-trifluoroethyl, 2-propynyl, sec-butyl, tert-butyl, and n-hexyl. 8.The method of claim 2 wherein R⁴ is H, R¹⁵ is H, R⁸ is n-propyl.